This invention relates to a pyruvate oxidase which is substantially free of ATP-ase and lactate oxidase Pyruvate oxidase is an enzyme which catalyzes the reaction of pyruvic acid, phosphate and oxygen to form acetylphosphate, carbon dioxide and hydrogen peroxide and has heretofore been derived from a strain of Lactobacillus delbruckii (Williams, F.R. & Hager, L.P. (1966) Arch. Biochem. Biophys., 116: 168-176), Pediococcus, Streptococcus or Aerococcus vilidans JP-A-58-40465.

Pyruvate oxidase is an oxidase on the substrate pyruvic acid, and hence it can be used for quantitative measurement of pyruvate in serum, further this enzyme can be used for the quantitative analysis of substrate which is related to various enzymes on pyruvate generating system such as glutamate - oxaloacetate transaminase, glutamate - pyruvate transaminase, lactate dehydrogenase or neuraminidase - N-acetylneuraminic acid aldorase, and the measurement of the enzyme activity of enzyme reaction systems which form pyruvates. So pyruvate oxidase is useful for a reagent in research and clinical diagnostics.

Quantitative determination of ADP using pyruvate oxidase has been known. Namely pyruvate is generated by an action of pyruvate kinase which forms ATP and pyruvate from ADP and phosphoenolpyruvate, and the thus generated pyruvate is oxidized by pyruvate oxidase from Pediococcus sp. B-0667, Streptococcus sp. B-0668 or Aerococcus vilidans IFO 12219 and IFO 12317 to form hydrogen peroxide which is measured. (Japan. Pat. Unexam. Publ. No. 59-15637). Also pyruvate oxidase from other origin has been knonw (ibid. No. 59-162877 and No. 59-159777).

Hitherto known pyruvate oxidase producing microorganims has disadvantages such as low productivity and contamination of other enzymes which cannot be removed, and hence high quality pyruvate oxidase can not be obtained.

Further detailed chemical structure of pyruvate oxidase, which catalyses a reaction from pyruvate, phosphate and oxygen to generate acetylphosphate, carbon dioxide and hydrogen peroxide, has not been reported.

A prior used pyruvate oxidase in an assay of ADP contains considerable amount of contaminant ATP-ase which resulted higher observation in blank. In an assay of ADP, phosphoenol pyruvate and pyruvate kinase is added in a specimen containing ADP to generate ATP and pyruvate, then the reaction mixture is reacted with pyruvate oxidase and generated H₂0₂ is measured. In this assay system contaminated ATP-ase catalyses a reaction;
ATP + H20 - ADP + Pi A resulted ADP is overlapped to the original ADP in a specimen. Therefore contaminant ATP-ase should be removed as much as possible. Furthermore prior pyruvate oxidase is contaminated with lactacte oxidase which resulted an error for measuring generated or consumed H₂0₂ in an assay using pyruvate oxidase. Therefore contaminate lactate oxidase should be removed.

We have succeeded to isolate pyruvate oxidase gene and to determine primary structure, and established the high produc tivity by applying genetic engineering technique.

The resulted pyruvate oxidase of the present invention has substantially no contaminant of ATP-ase with content below 0.0005%. The said pyruvate oxidase can be used for an assay of ADP with no increased color blank in assay. Further it does not contain lactate oxidase.

An object of the present invention is to provide a polydeoxyribonucleic acid which comprises:

• - being extraneous for the host, and
• - having base sequence coding an amino acid sequence of a polypeptide consisting of pyruvate oxidase which catalyses a reaction from pyruvate, phosphate and oxygen to generate acetylphosphate, carbon dioxide and hydrogen perioxide or an amino acid sequence including a polypeptide consisting of said pyruvate oxidase.

Another object of the present invention is to provide a polypeptide consisting of pyruvate oxidase which catalyses a reaction from pyruvate, phosphate and oxygen to generate acetylphosphate, carbon dioxide and hydrogen peroxide or a polypeptide including said pyruvate oxidase having an amino acid sequence from N-terminal of the formulawherein A is amino acid residue, hydrogen or acetyl and B is amino acid residue, -OH or -NH₂.

Further object of the present invention is to provide a process for production of pyruvate oxidase which comprises:

• - transforming a recombinant DNA into a host microorganism, in which the said recombinant DNA is contracted by inserting a polydeoxyribonucleic acid consisting of gene of pyruvate oxidase which catalyses a reaction from pyruvate, phosphate and oxygen to generate acetylphosphate, carbon dioxide and hydrogen peroxide or a polydeoxyribonucleic acid including the said gene, to obtain a transformant,
• - expressing a genetic information of said polydeoxyribonucleic acid by culturing said transformant, and
• - isolating a polypeptide consisting of pyruvate oxidase which catalyses a reaction from pyruvate, phosphate and oxygen to generate acetylphosphate, carbon dioxide and hydrogen peroxide, or a polypeptide including pyruvate oxidase.

More further object of the present invention is to provide a composition for assay comprising water soluble pyruvate oxidase which catalyses a reaction from pyruvate, phosphate and oxygen to generate acetylphosphate, carbon dioxide and hydrogen peroxide, with ATPase content of at least below 0.0005% and substantially containing no lactate oxidase activity.

Still further object of the present invention is to provide an assay method which comprises, in an assay of pyruvate in a specimen which contains pyruvate or generates pyruvate and at least contains lactate, treating said specimen with water soluble pyruvate oxidase which catalyses a reaction from pyruvate, phosphate and oxygen to generate acetylphosphate, carbon dioxide and hydrogen peroxide, with ATPase content of at least below 0.0005⁰/₀ and substantially containing no lactate oxidase activity, and measuring a consumed or generated composition.

Still more further object of the present invention is to provide an assay method of ADP which comprises at least including the following processes:

• (a) a process generating ATP and pyruvate from ADP and phosphoenol pyruvate by an action of transphosphorylation of pyruvate kinase,
• (b) a process forming detectable changes by generating acetylphosphate, carbon dioxide and hydrogen perioxide by an action of pyruvate oxidase on a substrate pyruvate using a composition of an effective ingredient consisting of water soluble pyruvate oxidase which catalyses a reaction from pyruvate, phosphate and oxygen to generate acetylphosphate, carbon dioxide and hydrogen peroxide, with ATPase content of at least below 0.005%, and
• (c) a process detecting the said detectable changes by acting enzyme and reagent.

Still another further object of the present invention is to provide an assay method of ATP which comprises at least including the following processes:

• (a) a process generating ADP and phosphate compound from ATP and non-phosphate compound by an action of transphosphorylation of kinase,
• (b) a process generating ATP and pyruvate form generated ADP and phosphenol pyruvate by an action of transphosphorylation of pyruvate kinase,
• (c) a process forming detectable changes by generating acetylphosphate, carbon dioxide and hydrogen peroxide by an action of pyruvate oxidase on a substrate pyruvate using a composition of an effective ingredient consisting of water soluble pyruvate oxidase which catalyses a reaction from pyruvate, phosphate and oxygen to generate acetylphosphate, carbon dioxide andhydrogen peroxide, with ATPase content of at least below 0.00050/c, and
• (d) a process detecting the said detectable changes by acting enzyme and reagent.

Still furthermore object of the present invention is to provide an assay method of non-phosphate compound which comprises at least including the following processes:

• (a) a process generating ADP and phosphate compound from ATP and non-phosphate compound by an action of transphosphorylation of kinase,
• (b) a process generating ATP and pyruvate form generated ADP and phosphoenoi pyruvate by an action of transphosphorylation of pyruvate kinase,
• (c) a process forming detectable changes by generating acetylphosphate, carbon dioxide and hydrogen peroxide by an action of pyruvate oxidase on a substrate pyruvate using a composition of an effective ingredient consisting of water soluble pyruvate oxidase which catalyses a reaction from pyruvate, phosphate and oxygen to generate acetylphosphate, carbon dioxide and hydrogen peroxide, with ATPase content of at least below 0.0005%, and
• (d) a process detecting the said detectable changes by acting enzyme and reagent.

In a polypeptide (I) amino acid residue A is one or more of amino acid residue, preferably hydrogen, Met or signal peptide. Example of B is acidamide or amino acid residue of more than one.

An example of a polydeoxyribonucleic acid of a gene consisting of pyruvate oxidase which catalyses a reaction from pyruvate, phosphate and oxygen to generate acetyl-phosphate, carbon dioxide and hydrogen peroxide or a polydeoxyribonucleic acid including said gene is at least a polydeoxyribonucleic acid containing gene of pyruvate oxidase which catalyses a reaction from pyruvate, phosphate and oxygen to generate acetylphosphate, carbon dioxide and hydrogen peroxide, and is a polýdeoxyribonucleic acid consisting of a base sequence coding an amino acid sequence of a polypeptide of pyruvate oxidase having a structure from N-terminal of the formula

In an amino acid sequence of polypeptide (II) of pyruvate oxidase, any polydeoxyribonucleic acid comprising any one of codons which codon corresponding to each amino acid can be mentioned, and preferably it can be a polydeoxyribonucleic acid having more than one codon except nonsence codon at 5'-terminal and/or more than one codon at 3'-terminal. For example a polydeoxyribonucleic acid having base sequence from 5'-terminal of the formulawherein X is a codon excepting TAA, TAG and TGA or hydrogen, and Y is codon or hyrogen can be mentioned.

In a base sequence (III), codon X can be a codon coding amino acid, and it can be a codon more than one which codes amino acid at 5'-terminal, and is preferably ATG or polydeoxyrebonucleic acid corresponding to signal peptide.

A codon Y can be a termination codon or amino acid coding codon, further it may have more than one codon which codes amino acid at 3'-terminal and is preferably to have a termination codon at 3'-terminal or plural codons.

A polydeoxyribonucleic acid comprising pyruvate oxidase gene, a polydeoxyribonucleic acid consisting of a gene having base sequence which codes amino acid sequence (II) or a polydeoxyribonucleic acid (III) can be obtained, for example, by isolating DNA from pyruvate oxidase producing microorganisms which is a donor of pyruvate oxidase gene, splitting the DNA by sonication or digstion with restriction enzyme, ligating the DNA with linear expression vector at the blunt end or cohesive end by DNA ligase, transferring the thus obtained recombinant DNA vector into host microorganism, obtaining microorganisms carrying the recombinant plasmid by selecting for the vector markers and activity of pyruvate oxidase, culturing the said microorganims, isolating and purifying the said recombinant DNA vector from the cultured cells, and isolating the polydeoxyribo-nucleic acid of pyruvate oxidase gene from the said recombinant DNA vector.

A microorganism which is a donor of pyruvate oxidase gene can be a pyruvate oxidase producing microorganism, for example, Lactobacillaceae and Streptococcaceae, such as Lactobacillus delbrucki [Japan. Pat. Unexam. Publ. No. 54-126791, No. 59-159777, No. 59-162877 and Arch. Biochem. Biophys., 116: 168 -176 (1966)]. Pediococcus sp., Streptococcus sp., Aerococcus viridans, Lactobacillus salivarius, Leuconostoc mesenteroides.

Also transformed microorganisms which is given pyruvate oxidase producing activity by means of recombinant DNA technology, can be used as a donor of pyruvate oxidase gene.

DNA can be obtained from gene donor microorganisms as follows. Microorganisms exemplified hereinabove are cultured for 1 - 3 days aerobically in a liquid medium. Cultured cells are collected by centrifugation and harvested cells are lysed to obtain lysate cells contianing pyruvate oxidase gene. Bacteriolysis can be made by treatment of cell wall lytic enzyme such a slysozyme or β-glucanase optionally treated with other enzymes such as protease or surface active agent such as sodium lauryl sulfate and other physical destruction means such as freeze-thawing or French press.

Isolation and purification of DNA from lytic cells can be made by combination of conventional means such as deproteination by phenol treatment, treatment with protease and/or, ribonuclease,alcohol precipitation or centrifugation.

Splitting of isolated or purified microbial DNA can be made by sonic treatment and restriction enzyme treatment, preferably restriction enzyme treatment for easyligation, especially using enzyme acting on specific nucleotide sequence, for example type II restriction enzyme such as EcoRl, Hindlll or BamHl. Preferable vectors are plasmid constructed for recombinant DNA technology from autonomically grown phage or plasmid in host cells.

Examples of phase are λgt • λC and λgt • λB for host cell of Escherichia coli.

Examples of plasmid are pBR322, pBR325, pACYC184, pUC12, pUC13, pUC18 or pUC19 for E. coli, pUB110 or pC194 for Bacillus subtillis, and shuttle vector for E. coli and Saccharomyces cerevisiae. It is preferable to obtain vector fragment by cleav ing the vector hereinabove with the same restriction enzyme used in cleavage of microbial DNA containing pyruvate oxidase gene hereinbefore.

Ligating method of microbial DNA fragment and vector fragment can be a conventional method using known _DNA ligase. For example after annealing a cohesive end of microbial DNA frag- ment a nd cohesive end of vector fragment, a recombinant DNA of microbial DNA fragment and vector fragment can be constructed byan action of preferable DNA ligase. If necessary, after anneal-ing, DNA is transforred into host mlcroorganlsms and a recombinant DNA can be prepared by means of in vivo DNA ligase.

Examples of host microorganism are preferably a microorganism in which recombinant DNA can be replicatable in stable and autonomically and transforred extraneous DNA can be expressed, for example Escherichia coli DH1, E. coli HB101, E. coli W3110 and E. coli C600.

As for a method of transfer of reoembinant DNA into a host microorganism, for example in case that the host cell is E. coli, a recombinant DNA can be transferred in the presence of calcium ion, and in case that the host cells is Bacillus, a competent cell method or protoplast method can be applied, further microinjection method may also be applied. The thus obtain and cultured transformant can produce large amount of pyruvate oxidase stably in a oxidase stably in a culture of nutrient medium.

Insertion of recombinant DNA can be selected by isolating the microorganisms which express drug resistant markers in the said vector and pyruvate oxidase production. For example micro-organisms which can grow in a selection medium for drug resistance and have activity of pyruvate oxidase production, is preferably selected. ,

Selected recombinant DNA consisting pyruvate oxidase gene is isolated from transformant cells and is easily transform the other host cells. Also pyruvate oxidase gene consisting of DNA is digested by restriction enzyme to obtain DNA fragment of pyruvate oxidase gene, which is ligated with vector fragment obtained by the same way, and the ligated DNA can easily be transferred Into the host cells.

DNA coding pyruvate oxidase protein which shows substantial pyruvate oxidase activity, is an artificial mutant gene derived from pyruvate oxidase gene of the present invention by means of genetic engineering technique and this mutant gene which is prepared by the methods hereinbefore is inserted into a vector to construct recombinant DNA, then pyruvate oxidase protein can be produced.

A base sequence of the thus obtained pyruvate oxidase gene is determined by dideoxy method (Science, 214: 1205 -1210, 1981) and amino acid sequence of pyruvate oxidase Is predicted by the base sequence. A partial amino acid sequence of N-terminal of pyruvate oxidase peptide Is determined by the following method. Pyruvate oxidase producing microorganisms which is a donor of pyruvate oxidase gene is cultured in a nutrient medium to accumulate pyruvate oxidase endogeneously. The cultured cells are collected by filtration or centrifugal means, disrupted by means of mechanically or enzymatically with lysozyme, solubilized by adding EDTA and/or surface active agent and isolated the enzyme solution. Aqueous solution of pyruvate oxidase is treated by ammonium sulfate fractionation, gel-filtration, adsorption chrbmatography or ion-exchange chromatography with or without concentration to obtain highly purified pyruvate oxidase. A partial sequence of amino acid of N-terminal of pyruvate oxidase is determined by the liquid phase protein sequencer (Beckman System 890 ME) and confirmed the identity as compared with that of pyruvate oxidase obtained by gene tic manipulation.

Cultivation condition of the transformant is determined by considering nutrient physiological properties of cells, and is usually made by conventional liquid culture and submerged aeration culture ls preferable for industrial production.

A conventional medium for culturing microorganisms can preferably be used. For the carbon sources, assimilable carbon sources such as glucose, sucrose, lactose, maltose, fructose, molasses, pyruvic acid or the like can preferably be used. Assimilable nitrogen sources such as peptone, meat extract, yeast extract, casein hydrolyzate or the like can be used. Various inorganic salts such as phosphates, carbonates, sulfates, salts of magnesium, calcium, potassium, divalent iron, manganese or zinc or specific amino acids and vitamins can be used.

The culturing temperature can be selected within the range for growth of microbial cells and production of pyruvate oxidase, and is preferably 20-42°C for E. coli. The culturing time can be altered depending on conditions and is terminated when the pyruvate oxidase production Is substantially complete, and is usually 12-48 hours.

The pH of the medium can be altered depending on conditions for growing cells and producing pyruvate oxidase, and is usually pH 6.0-8.0.

To separate pyruvate oxidase from the culture, the cultured mass is filtered or centrifuged to collect the cells, which are disrupted by treatment with mechanical means or enzymes such as lysozyme or in case that enzyme is exogeneously existed in a medium, cultured mass is filtered or centrifuged to separate culture filtrate. Further if necessary pyruvate oxidase is solubilized by adding EDTA and a surfactant to extract the enzyme. The thus-obtained solution of pyruvate oxidase is treated with or without concentration, and thereafter the enzyme is precipitated by salting out with the addition of a soluble salt such as ammonium sulfate or sodium chloride. Low molecular weight impurities are removed by dialysis. Furthermore impurities in the solution of pyruvate oxidase are preferably removed by adsorption chromatography, ion-exchange chromatography or gel filtration. The enzyme solution thus obtained is treated by vacuum concentration and lyophilization to produce powdered pyruvate oxidase.

Abbreviations of amino acid, peptide, nucleic acid, nucleic acid related substance and others are as follows.

All the amino acid shows L-form.

• DNA: deoxyribonucleic acid
• RNA: ribonucleic acid
• A: adenine
• T: thymine
• G: guanine
• C: cytocine
• Ala: alanine
• Arg: arginine
• Asn: asparagine
• Asp: aspartate
• Cys: cysteine
• Gln: glutamine
• Glu: glutamate
• Gly: glycine
• His: histidine
• Ile: isoleucine
• Leu: leucine
• Lys: lysine
• Met: methionine
• Phe: phenylalanine
• Pro: proline
• Ser: serine
• Thr: threonine
• Trp: tryptophane
• Tyr: tyrosine
• Val: valine

Pyruvate oxidase produced by the present invention has the following physico-chemical properties.

• (1) Enzyme action:
  • The enzyme catalyzes the oxidative reaction of pyruvic acid, inorganic phosphate and oxygen to form acetylphosphate, carbon dioxide and hydrogen peroxide:
  • CH₃COCOOH + HOPO₃^-- +O₂→CH₃COOPO₃^-- +CO₂+H₂O₂
• (2) Substrate specificity: Specific for pyruvate.
  • No action on a-ketoglutarate, oxaloacetate, lactate, acetate and alanine.
• (3) Isoelectric point: pH 4
• (4) Molecularweight: 150,000 - 155,000 (gel-filtration method)
• (5) Optimum pH : 6.5 - 7.5
• (6) pH-stability: 5.5 - 7
• (7) Lactate oxidase activity:
  • Substantially no lactate oxidase activity is observed. Activity of lactate oxidase is measured according to Japan. Pat. Unexam. Pat. Publ. No. 55-76.
• (8) ATP-ase activity:
  • Contaminant ATP-ase activity is observed less than 0.0005%.

• (9) Nature: soluble in water.
• (10) Amino acid sequence: shown as formula (I).

The assay method of pyruvate oxidase of the present invention uses a reaction mixture as follows: 0.5 M potassium pyruvate 0.1 ml

• 0.5 M phosphate buffer (pH 7.0) 0.2 ml
• 0.2% 4-aminoantipyrin 0.1 ml
• 0.2% N,N-dimethylaniline 0.2 ml
• 10 mM MgC1₂ 50 µl
• 10 mM thiaminepyrophosphate 20 µl
• peroxidase (45 U/ml) 0.1 ml
• 1 mM FAD 10 µl
• distilled water 0.22 ml

The above reaction mixture (1.0 ml) is pre-incubated at 37°C for 3 minutes. To this solution is added the enzyme solution (20 µl) and incubated at 37° C for 10 minutes. 0.1 M citrate buffer (pH 6.0, 2 ml) containing 0.1 M EDTA is added to stop the reaction. The violet color formed is measured by colorimetric method at 565 nm.

A unit (1 unit, 1 U) of enzyme activity is defined as the activity which generates 1 µmole of hydrogen peroxide per minute.

Fundamental reactions of assay method using pyruvate oxidase are illustrated as follows.

In the assay, the following reaction can at least be included and various combination of known prior reactions therefor can be used.

• ① a reaction of glutamate-pyruvate-transaminase: alanine + α-ketoglutarate→ pyruvate + glutamate
• ② a reaction of glutamate-oxaloacetate-transaminase and oxaloacetate decarboxylase: aspatate + a-ketoglutarate → oxaloacetate + glutamate oxaloacetate → pyruvate + C0₂
• ③ a reaction of pyruvate kinase: ADP + phosphoenolpyruvate→ATP + pyruvate
• ④ a reaction of N-acetyleneulaminate aldolase: N-acetyleneulaminate + H₂O→ N-acetylmanosamine + pyruvate
• ⑤ a reaction of lactate dehydrogenase: lactate + NAD - pyruvate + reduced NAD

Preferable combination for the above reaction is, for example, ammonium generated from urea by an action of urease, ammonium generated from creatinine by an action of creatinine diaminase, or ammonium liberated from the other reaction system, is reacted with glutamate and ATP in the presence of glutamine synthetase to form ADP, glutamine and inorganic phosphate, and the thus generated ADP is combined with ADP assay system using pyruvate kinase and phosphoenolpyruvate hereinabove.

pyruvate kinase oyruvate oxidase (c) measuring consumed 0₂ or generated H₂0₂ or C0₂

Various assay can be made by combining prior reaction systems to the above processes.

kinase (d) measuring consumed 0₂ or generated H₂0₂ or C0₂

The same as of the above III.

Examples of ADP generating or consuming enzyme reaction system are illustrated as follows.

ADP generating reaction:

• ① a reaction of hexokinase; ATP + D-hexose→ADP + D-hexose-6-phosphate
• ②a reaction of glucokinase; ATP + D-glucose→ADP + D-glucose-6-phosphate
• ③ a reaction of amylase (with maltase);
  glucose polymer (soluble starch, amylose or other oligosaccharide or derivatives thereof) + nH20 D-glucose + maltose, and maltose + 2H₂O→ 2D-glucose ATP + D-glucose-ADP + D-glucose-6-phosphate
• ④ a reaction of adenosine kinase; ATP + adenosine→ADP + AMP
• ⑤ a reaction of thymidine kinase; ATP + thymidine→ADP + thymidine-5'-phosphate
• ⑥ a reaction of NAD kinase; ATP + NAD+ -+ADP + NADP+
• ⑦ a reaction of enzyme action from NADH + H⁺ to generate NAD⁺ NADH+H⁺ + substrate A (oxydized form)→ NAD⁺ + H₂A and ATP+NAD+→ADP+NADP⁺
• ⑧ a reaction of riboflavine kinase; ATP + riboflavin→ADP + flavin-5'-phosphate
• ⑨ a reaction of glycerol kinase; ATP + glycerol→ADP + glycerol-3-phosphate
• ⑩ a reaction of triglyceride assay (with lipase); triglyceride + 3H20 - glycerol + 3 fatty acid, and ATP + glycerol→ADP + glycerol-3-phosphate
• ⑪ a reaction of lipase assay; di- or triglyceride + nH₂O→ glycerol + n fatty acid, and ATP + glycerol→ ADP + glycerol-3-phosphate
• a a reaction of choline kinase; ATP + choline→ADP + choline phosphate
• ⑬ a reaction of choline esterase assay ; choline ester (fatty acid ester or aryl ester; RCOO) + H₂O→ choline + RCOO^- and ATP + choline→ADP + choline phosphate
• ⑭ a reaction of phospholipid (lecithin) assay (with phospholipase D); lecithin + H₂O→ phosphatidate + choline ATP + choline→ ADP + choline phosphate
• ⑮ a reaction of protein kinase assay; ATP + protein→ADP + phospho-protein
• ⑯ an assay of creatine kinase or creatine; ATP + creatine - ADP + creatine phosphate

ATP generating reaction (ADP-consuming reaction):

• ① a reaction of carbamate kinase assay, ADP or carbamoyl-phosphate assay; ADP + carbamoyl phosphate→ NH₃ + ATP + C0₂
• @ an assay of phosphoglycerate kinase activity or ADP; ADP + D-1,3-bisphosphoglycerate→ATP + 3-glycerol-D-glycetrate
• ③ a reaction of formate kinase assay, ADP orformyl-phosphate; ADP + formyl phosphate - ATP + formate
• ④ a reaction of creatine kinase assay, ADP or creatine-phosphate assau; ADP + creatine phosphate - ATP + creatine
• ⑤ an assay of ammonia kinase activity or ADP; ADP + phosphoramide→ATP + NH₄⁺
• ⑥ an assay of myokinase activity or ADP; 2ADP→ATP + AMP

An assay method of generated ADP or consumed ADP is illustrated in Japanese Patent Appln. No. 53-86350 "assay kit and method using pyruvate oxidase".

In order to activate the pyruvate oxidase reaction system, FAD, thiaminepyrophosphate and phosphate are added. Further for activation of the enzyme, an ion-liberating salt which liberates calcium ions, cobalt ions, magnesium ions or manganese ions, in the form of chloride is preferably added thereto. An indicator such as a coloring indicator or fluorescent indicator for hydrogen peroxide can preferably be used.

The amount and ratio of components in the enzyme reaction system can be selected for substantial enzyme reaction and will be varied according to the amount of pyruvate, temperature and time of enzyme reaction. For example, 1-200 U of pyruvate oxidase can preferably be used.

Pyruvate oxidase can be in a microcapsulated form or in an immobilized form of covalent linkage with an organic or inorganic carrier or adsorbed on a carrier.

Further 0.1 - 20 nmoles of FAD, 0.05-0.5 µmole of thiaminepyrophosphate, 1-10 µmoles of inorganic phosphate and 0.05-0.1 µmoles of ion liberating salt per test can preferably be used.

The molar ratio of indicator for hydrogen peroxide is at least an equimolar or excess amount of generated hydrogen peroxide. In the case of the peroxidase, 0.5-20 U per test is preferably used. These components of the enzymatic reaction mixture are preferably used by dissolving in the buffer of suitably adjusted pH. The thus-prepared enzymatic reaction system is used for the analysis of pyruvic acid, ADP, ATP or related non-phosphate compound.

Assay is performed by incubation with the sample and a reagent mixture. The reagent mixture is preferably a kit of necessary reagents. For assaying, consumed component or generated component is measured. Measuring the amount of ocygen consumption by dissolved oxygen meter is preferable as an assay method. In this case no indicator for hydrogen peroxide is necessary. As for the assaying of a generated component, measurement of the amount of hydrogen peroxide is preferable, for example by a hydrogen peroxide electrode meter such as YSI-oxidase meter or by colorimetric or fluorometric assay with an indicator for hydrogen peroxide. The assay can be performed preferably for 10-60 minutes and at 20-40° C, preferably at 35-37° C. The indicator for hydrogen peroxide is a combination of one or more chromogen or fluorescents, which is effected by coupling with hydrogen peroxide. Examples of such Indicators are combinations fo tetravalent titanium compounds and xylenol orange which couples with hydrogen peroxide to form a stabfe red color, or a combination of phenol or N,N-dimethylaniline or homovanillic acid, 4-aminoantipyrine and peroxidase for measuring color or fluorescence. 4-aminoanitipyrin can be replaced by 4-aminophenazone. A combination of 2,6-dichlorophenol indophenol and peroxidase and of guaiacol and peroxidase can also be used. The indicator can be previously prepared as a solution.

The amount of pyruvic acid can be measured by calculation from corresponding standard curves of consumed oxygen or generated hydrogen peroxide.

Colorimetric or fluorometric assy is performed by measuring the absorption at a suitable wave length such as 565 nm.

Phosphate as a consumed component or acetylphosphate as a generated component can also be assayed by any conventional method.

Pyruvate oxidase of the present invention does not contaminated with lactate oxidase and content of ATP-ase is less than 0.0005%, and hence is advantageous as compared with prior known pyruvate oxidase which contains lactate oxidase and 0.005% of ATP-ase.

ATP-ase assay method and coloring reagent for ADP assay are illustrated as follows..

1. ATP-ase assay:

Reagent 1 (1.0 ml) (containing 40 U pyruvate oxidase) is preincubated at 37° C for 1 hour. ADP reagent 2 (1.0 ml) is added thereto, and incubated at 37°C for 15 min., further added 100/0 SDS (sodium dodecyl sulfate) (1.0 ml) to dissolve turbidity, and measured at 550 nm.

ΔA₅₅₀ nm : In the reagent 1, ATP is deleted. Incubation was carried as same way and absorbancy was deducted.

• 18 : molecular absorbancy coefficient
• 0.1 : enzyme solution (1 ml)
• 3.0 : total reaction volume (ml)
• 60 : reaction time (min.)

400 : pyruvate oxidase activity (400 u/ml).

Following examples illustrate the present invention but are not construed as limiting.

Chromosomal DNA of Aerococcus vilidans IFO 012219 was isolated by following procedure.

The strain was cultured overnight with shaking in a bouillon medium (150 ml, containing 0.5% sodium thiosulfate) at 37° C. Cultured cells were collected by centrifugation at 3,000 rpm for 10 min. Lysozyme solution (10 mg/ml, 1 ml) was added to a suspension of the cells in a solution of 10% sucrose, 50 mM Tris-HCI (pH 8.0) and 50 mM EDTA, and incubated at 37°C for 15 min., further added 10% SDS (sodium dodecyl sulfate) (1 ml). Equal volume of a mixture of chloroform-phenol (1:1) was added to the suspension, mixed with stirring, centrifuged at 10,000 rpm for 3 min. to separate aqueous layer and solvent layer. Aqueous layer was collected. Two-fold in volume of ethanol was gently added to the aqueous layer to make double phase, and DNA was collected by winding on a glass rod with gentle stirring. Collected DNA was dissolved in a solution (10 ml) consisting of 10 mM Tris+HCI (pH 8.0) and 1 mM EDTA (hereinafter designated as TE solution). A solution was treated with an equal volume of a mixture of chloroform - phenol and centrifuged to separate an aqueous layer. Two-fold in volume of ethanol was added to the aqueous layer and DNA was isolated by the same way as above, then dissolved in the TE solution (2 ml).

A strain of Escherichia coli pM 191 [J. Bacteriol., 134:1141 (1981), ATCC 37033] carrying pACYC 184 was cultured with shaking in BHI medium (Difco, 1 lit.). When growth of bacterial cells measured by turbidity was observed as OD₆₆₀ = 1.0 spectinomycin (final concentration 300 µg/ml, plasmid resistant marker is chloramphenicol) was added, and continued to shake at 37°C for 16 hours. Grown cells were collected at 3,000 rpm for 10 min., and a plasmid DNA was prepared according to the method of lysozyme - SDS and cesium chloride - ethidium bromide [Maniortis et al., Molecular Cloning, pp 86-94, Cold Spring Harbor (1982)].

(i) Chromosomal DNA (2 µl, approximately 0.5 µg) of A. vilidans prepared in Example 1 was mixed with EcoRl cleavage buffer (1 µl) (500 mM Tris-HCI, pH 7.5, 70 ml MgCl₂, 1 M NaCl and 70 mM mercaptoethanol), E CoRI (1 µl:Takara Syuzo Co., Ltd) and water (6 µl), and cleaved at 37°C for 1 hour. Separately prepared plasmid pACYC 184 DNA (ca 0.3 µg) was cleaved in the same way with EcoRl. Alkaline phosphatase (hereinafter designated as BAP, Takara Shuzo Co.) (0.6 unit) was added thereto and incubated at 65° C for 1 hour. The EcoRl-treated two kinds of DNA were mixed, 3M sodium acetate added (1/10 volume), the mixture further treated with an equal volume of a mixture of chloroform and phenol, and the aqueous layer was collected by centrifugation.

Two-fold in volume of ethanol was added to the aqueous layer, which was centrifuged to precipitate the DNA which was dried in vacuo. Lyophilized DNA was dissolved in water (89 µl). Ten times conc. ligation buffer (0.5 M Tris-HCI, pH 7.6, 0.1 M MgCI₂, 0.1 M dithiothreitol, 10 mM spermidine and 10 mM ATP) (10 µl) and T4 DNA ligase (1 µl, Takara Shuzo Co., 175 units) were added thereto, mixed and let stand at 4°C for overnight. The said DNA solution was treated with chloroform - phenol, DNA collected by precipitation with ethanol,dried in vacuo and dissolved in TE (10 µl).

(ii) Logarithmically growing Escerichia coli W3110 (obtained from National Institute of Heredity, Japan, stock No. ME 7778, ATCC 27325) in BHI medium (100 ml, Brain Heart Infusion, Difco) was collected by centrifugation at 10,000 rpm for 2 min., and suspended in an ice cold solution (40 ml, pH 5.8) consisting of 30 mM potassium acetate,100 mM RbCl,10 mM CaCl₂, 50 mM MnCl₂ and 15% glycerine. The suspension was let stand for 5 min. at 0°C, centrifuged and discarded the supernatant. Further precipitate was suspended in a solution (4 ml, pH 6.5) consisting of 10 mM MOPS buffer (Dotite Co.), 75 mM CaCl₂, 10 mM RbCI and 150/0 glycerine, and let stand for 15 min, at 0°C to prepare competent cells.

(iii) DNA solution (10 µl) prepared as described in (i) was added to the E. coli suspension from (ii) (200µl) and let stand for 30 min. at 0°C. BHI medium (1 ml) was added thereto, kept at 37°C for 90 min. and 100 µl thereof was spread over BHI agar plate containing tetracycline (15 µg/ml), then cultured at 37°C overnight to obtain a transformant. The transformant cells were replicated on a POP medium plate (peptone 5 g, meat extract 2 g, yeast extract 5 g, NaCl 1 g, K₂HP0₄ 1 g, MgS0₄ 0.5 g, peroxidase 500 IU, FAD 7.85 mg, dianisidine 0.1 g, thiaminepyrophosphate 42.4 mg, pyruvate 1ℓ, agar 15 g, distilled water 1 lit., pH 7.0) and further cultured at 37° C overnight. Approximately 4,500 colonies of transformant were checked, and one colony was obtained the surrounding of which was brownish brown. This strain was designated as Escherichia coli W3110-pOXl3 and was deposited at FERM P-No. 9071 (FERM BP-1565).

The strain when cultured at 37°C overnight in BHI medium was shown to yield 3 units/ml of pyruvate oxidase.

A plasmid in this strain was isolated according to the method in Example 2 and was designated as pOX13 which contains pyruvate oxidase gene and pACYC 184 gene.

a pOX13 plasmid DNA was prepared from E. coli W3110 pOX13 according to the same procedure as described in Example 2 for the preparation of pACYC 184.

A restriction map of pOX13 DNA by digestion with restriction enzymes Clal, EcoRV, Hindlll, Scal, Pstl, Pvull, Xbal (Takara Shuzo Co.) and Hpal (Toyobo Co.) was prepared with the result shown in Fig. 1.

Base sequence of DNA including pyruvate oxidase gene was determined by dideoxy method (Science, 214: 1205-1210,1981) using M13 phage. In Fig. 2-1, 2-2 and 2-3, the base sequence of POP structural gene and the amino acid sequence of the polypeptide for which the gene codes are illustrated.

E. coli W3110 pOXI3 was cultured in BHI medium (Difco) (20 lit.) at 37°C for 18 hours in a jar-fermenter, and centrifuged at 5,000 rpm for 10 min. to collect the cells. Bacterial cells were washed with physiological saline (2 lit.) and suspended in 10 mM phosphate buffer (pH 7.0). Lysozyme (1 mg/ml), EDTA.2Na (2 mM) and Triton X-100 (0.1%) were added and kept and 37° C for 30 min. then centrifuged at 5,000 rpm for 10 min. to separate the supernatant solution.

Ammonium sulfate was added to the supernatant (1.9 ) at 40~66% saturation and the precipitate was collected by centrifugation (5,000 rpm, 10 min.). The precipitate was dissolved in 10 mM phosphate buffer (pH 7.0, containing 10 µMFAD) (200 ml), then desalted by Sephadex G-25. The desalted enzyme solution was charged on a column of DEAE-Sepharose CL-6B for ion exchange chromatography and the active fraction was collected, desalted and lyophilized to obtain an enzyme powder. The enzyme shows 51 µ/mg of activity.

Amino acid sequence of 10 amino acids from N-terminal of pyruvate oxidase obtained in Example 5 was determined by amino acid sequencer (Beckman System 980 ME).

Approximately 80% thereof contains Met at the N-terminal and the sequence from amino acid position 1 is shown as "Ser, Asp, Asn, Lys, Ile, Asn, Ile, Gly, Leu, (Ala)".

Blank value of reagent in pyruvate oxidase with contaminant ATP-ase using ADP coloring reagent prepared in (2) hereinbefore is observed. As shown in Fig. 3, an increase in the blank value caused by generating ADP from ATP by contaminant ATP-ase was observed with known pyruvate oxidase. No Increase in the blank value was observed in pyruvate oxidase of the present invention with contaminant ATP-ase of 0.0005%.

Reagents of the following composition containing pyruvate oxidase with contaminant ATP-ase of 0.005% and 0.00050/₀ were prepared, and triglyceride in serum was comparatively measured by using the reagent.

0.2 M PIPES-NaOH (pH 7.3) 0.3 ml 0.1 M ATP 0.15 ml 0.1 M MgC1₂ 0.15 ml 10 mM thiaminepyrophosphate 0.3 ml 0.3% TOOS 0.3 ml 0.2% 4-aminoantipyrine 0.3 ml peroxidase (45 u/ml) 0.3 ml glycerokinase (25 u/ml) 0.05 ml 1 mM FAD 0.05 ml 10 mM phosphate buffer (pH 7.3) 0.15 ml pyruvate oxidase (500 u/ml) 0.03 ml 0.1 M phosphoenolpyruvate 0.03 ml pyruvate kinase (500 u/ml) 0.03 ml lipase (3,000 u/ml) 0.03 ml H₂0 0.56 ml

Human serum (20-100 µl) was added to the triglyceride coloring reagent hereinabove (3.0 ml), which is either one immediately after preparation or the other stored for 20 hours at room temperature in the dark room. The mixture was incubated at 37° C for 15 min. then immediately measured the absorbance at 550 nm. Result is shown in Fig. 4.

In the figure:

• •-•: immediately after preparation of the reagent with pyruvate oxidase of contaminant 0.005% ATP-ase.
• •--•: immediately after preparation of the reagent with pyruvatge oxidase of contaminant 0.0005% ATP-ase. ○-○: 20 hours stored reagent with pyruvate oxidase of contaminant 0.005⁰/₀ ATP-ase. 0--0: 20 hours stored reagent with pyruvate oxidase of contaminant 0.00050/o ATP-ase.

Reagents of the following composition containing pyruvate oxidase with contaminant ATP-ase of 0.005% and 0.0005% were prepared, and hexokinase activity is measured.

0.2 M HEPES-NaOH (pH 7.6) 0.3 ml 0.1 M ATP 0.15 ml 2 M glucose 0.30 ml 0.1 M MgCl₂ 0.24 ml 10 mM thiaminepyrophosphate 0.30 ml 0.30/o TOOS 0.30 ml 0.20/o 4-aminoantipyrine 0.30 ml peroxidase (45 u/ml) 0.30 ml 10 mM phosphate buffer (pH 7.6) 0.15 ml pyruvate oxidase (500 u/ml) 0.30 ml 0.1 M phosphoenolpyruvate 0.03 ml pyruvate kinase (500 u/ml) 0.03 ml H₂0 0.57 ml

Hexokinase (Oriental Yeast Co.) (50 µl) was added to the reagent for hexokinase asay immediately after preparation or 20 hours stored in dark place at room temperature, and continuously measured the absorbancy at 550 nm at 37° C.

Hexokinase activity was calculated by an increase absorption per minute after 3 min. of enzyme addition. (Fig. 5)

In Fig. 5:

• •-•: immediately after preparation of the reagent with pyruvate oxidase of contaminant 0,005% ATP-ase.
• •--•: immediately after preparation of the reagent with pyruvate oxidase of contaminant 0.0005% ATP-ase. 0-0: 20 hours stored reagent with pyruvate oxidase of contaminant 0.005% ATP-ase. 0--0: 20 hours stored reagent with pyruvate oxidase of contaminant 0.0005% ATP-ase.

As shown hereinabove a reagent for ADP assay prepared by adding pyruvate oxidase of contaminant less than 0.0005% provides low increased blank value of coloring reagent and hence long term storage of the reagent can be achieved.

Human serum (20 µl, three types of sample A, B and C) was added to the reagent for GOT activity assay (1.0 ml) of the following composition, and incubated at 37° C for 30 min. Reaction ws stopped by adding Mcllvain buffer (pH 5.5, 2.0 ml) containing 1% Triton X-100 and 0.1 M EDTA and the absorbance at 550 nm was measured. Reaction solutions without adding L-aspartate and a-ketoglutarate were prepared and treated as above for blank assay.

0.5 M HEPES-NaOH (pH 7.0) 0.08 m! 0.2 M KH₂P0₄-NaOH (pH 7.0) 0.02 ml 0.2 M L-aspartate (pH 7.0) 0.5 mi 0.2 M a-ketoglutarate (pH 7.0) 0.05 ml 20 mM thiaminepyrophosphate 0.05 ml 0.5 M MgCl₂ 0.01 ml 20 mM 4-aminoantipyrine 0.05 ml 20 mM TOOS 0.05 ml peroxidase (45 u/ml) 0.05 ml pyruvate oxidase (160 u/ml) 0.05ml oxaloacetate decarboxylase 0.05 ml H₂0 0.04 ml

As shown in the result, an increase in the absorbance in blank assay was observed for using prior known pyruvate oxidase due to serum lactate interference, and so difference in absorbance should carefully be determined. On the contrary, no increase in blank assay was observed for using pyruvate oxidase of the present invention, and so pyruvate oxidase of the present invention is not contaminated with lactate oxidase and can be used without calculating the difference in absorbance between assay and blank-value. According to the present invention, base sequence of pyruvate oxidase gene and amino acid sequence of pyruvate oxidase were provided. Also a process for production of pyruvate oxidase applying genetic engineering technique was provided.

Further, assay method using pyruvate oxidase of the present invention shows almost no increase in blank-value and so the long term storage of reagent can be achieved.